CRISPR technology highlights genes that contribute to the development of emphysema and COPD

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Overview of the CRISPRi platform for COPD GWAS gene interrogation. (A) Gene editing strategy to generate inducible CRISPRi iPSC/ESC lines with NKX2-1–GFP and/or SFTPC-tdTomato reporter. (B) Schematic representation of dox-inducible expression of dCas9-KRAB and lentivirus-delivered gRNA targeting the transcription start site (TSS) of genes of interest (GOI) to mediate knockdown. (C) Effector genes at genome-wide significant COPD loci were identified using gene expression, methylation status, coding associations, deoxyribonuclease (DNase) hypersensitivity, chromatin interactions, and/or gene set similarity as described in -above (11). Genes were further filtered based on expression during lung-directed differentiation of iPSCs, expression in differentiated iAT2s, and expression changes during human lung development. The expression levels of the final selected genes, FAM13A, DSP, TGFB2, MFAP2, RBMS3, SOX4, SOX9, HHIP and ADGRG6, are shown in iPSCs, iPSC-lung progenitors, early (16 to 17.5 weeks of gestation) and late ( 20 to 21 weeks of gestation) human fetal lung (HFL), iAT2s grown as 3D alveolospheres or at the air-liquid interface (ALI), primary pediatric (13-month-old male donor) and adult (32-year-old male donor) AT2s , grown in CK + DCI with MRC-5 cells, and freshly isolated primary adult AT2s. credit: Scientific progress (2022). DOI: 10.1126/sciadv.abo6566

Researchers at the Center for Regenerative Medicine at Boston Medical Center and Boston University School of Medicine have used CRISPR variants to understand the functions of genes that cause emphysema and chronic obstructive pulmonary disease (COPD). Posted in Scientific progressresearchers discovered functional consequences by turning off gene expression that contributes to the pathogenesis of these diseases.

“This is the first time CRISPRi and CRISPRa have been applied to human induced pluripotent stem cells to understand the functional role of these genes,” said Andrew Wilson, MD, a pulmonologist at Boston Medical Center and associate professor of medicine at Boston University School of Medicine. medicine. “This brings us closer to understanding how genetic factors contribute to susceptibility to emphysema.”

COPD and emphysema are the third leading cause of death worldwide, creating a significant disease burden. Emphysema is a complex genetic disease caused by a mutation or variant in a number of genes that contribute to making some individuals more susceptible to the disease than others. Genome-wide association studies (GWAS) have included variants in or near a growing number of genes, but understanding of their functions and how they potentially contribute to the development of COPD and emphysema is quite limited.

“No significant new pharmacologic agents have been developed to help treat the large number of patients suffering from COPD or emphysema worldwide,” said Dr. Rhiannon Werder, a postdoctoral fellow at Boston Medical Center’s Center for Regenerative Medicine and Boston University School of Medicine. . “We hope this study will help understand the genetics of the disease, improve our understanding of how the disease manifests at the cellular level, and help develop new therapies to treat these conditions.”

The researchers created a system using CRISPR variants to turn off either expression of a gene of interest using CRISPR interference (CRISPRi) or overexpression of a gene of interest using CRISPR activation (CRISPRa) in induced pluripotent stem cells (iPSCs). The researchers grew these cells in a dish and differentiated them to generate cells that are found in the lung. The cell type studied is called alveolar epithelial cell type 2, the progenitor cell for the alveolus – the alveolus is the part of the lung where gas exchange takes place and is the structure that is damaged in emphysema. So by understanding how GWAS genes affect type 2 cells, researchers can begin to understand how they may contribute to diseases that affect these cells, such as emphysema.

After the type 2 cells were generated, the researchers used CRISPRi to turn off the expression of nine different GWAS genes and analyzed them to see how the cells were affected, particularly their ability to proliferate, something they should be able to do in response to injury similar to what occurs in emphysema. The researchers noticed that turning off one particular gene, desmoplakin (DSP), caused the cells to increase their proliferation and increased the expression of genes associated with cell maturation. The researchers found that cells in which DSP expression was turned off prior to smoke exposure turned off the expression of cell junction genes to a greater extent than in controls. They were also better at forming new colonies, a measure of progenitor function, than controls. The researchers then examined mice in which DSP had been deleted from their lung epithelial cells, compared to control mice with normal DSP. The researchers found that type 2 cells in the DSP deletion mice were more proliferative after injury, consistent with findings in human type 2 iPSC cells.

DSP appears to modulate the proliferative capacity of type 2 cells at baseline and after injuries that are associated with human disease, such as smoke exposure. Lower levels of DSP expression increase the proliferative capacity of type 2 cells in the system, potentially making them more capable of responding to injury. Conversely, the higher expression levels found in cells harboring the variant associated with COPD risk by GWAS appear to make the cells less proliferative after smoke exposure, potentially explaining how this gene contributes to the disease.

The team identified changes in gene expression and cellular interactions involved in COPD

More info:
Rhiannon B. Werder et al, CRISPR interference query of COPD GWAS genes reveals functional significance of desmoplakin in iPSC-derived alveolar epithelial cells, Scientific progress (2022). DOI: 10.1126/sciadv.abo6566

Courtesy of Boston Medical Center

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